Nucleic acids as a biotechnological product are becoming increasingly important since they are often used as a therapeutic substance for the treatment of diseases (i.e. gene therapy) or as a means of protection from infectious diseases (i.e. genetic immunization). In the field of non-viral gene transfer, the genetic information contained within the polynucleotide is typically transferred into the target cell as a vector, either in a pure form (“naked DNA”) or alternatively as a complex, e.g. associated with other substances (including lipids, liposomes, PEI, carboplexes, proteins, gold particles, vesicles, nano-containments or magnetic beads) to either protect the nucleic acid or release it in a particular way or to target the nucleic acid to specific cells, where the gene expression should occur.
For the production of the nucleic acids, standard microorganisms are typically used that replicate extra-chromosomal circular nucleic acids. Common examples of such nucleic acids include plasmids, cosmids, BACs (bacterial artificial chromosomes), YACs (yeast artificial chromosomes) or MACs (mammalian artificial chromosomes).
The microorganisms are then cultivated to a high cell density with a maximum yield of the desired nucleic acid. In some applications, the desired extra-chromosomal nucleic acid might be a modified product (e.g. a mini-circle deriving from a plasmid vector, known collectively as “nucleic acid” or “plasmid”). For the isolation of the nucleic acid, the cells have to be broken up (e.g. disrupted, extracted, lysed). For this purpose, different physical and chemical processes are known. The major goal of such processes is to obtain a high yield of nucleic acid and to have the capacity to perform any subsequent purification steps using the simplest means possible.
For the isolation of plasmid DNA, alkaline lysis, a well-known chemical extraction procedure is usually applied (see Birnboim, H. C., Doly, J. (1979), A Rapid Alkaline Extraction Procedure for Screening Recombinant Plasmid DNA, Nucl. Acid. Res. 7, 1513-1523). In this procedure, suspended cells are lysed using an alkaline extraction reagent. During the neutralization step, the lysate is neutralized with an acidic potassium acetate solution, whereby proteins, chromosomal DNA and cell debris flocculate together with potassium dodecyl sulphate. Plasmid DNA (i.e. nucleic acid) remains within the resulting solution and can be separated from the majority of the flocculated contaminants.
The use of nucleic acids in pharmaceutical or technical applications (e.g. gene therapy, nucleic acid vaccination, or product labeling) requires a manufactured yield in gram and kg amounts; in the case of market supply, even larger scale production is required. For laboratory use and pilot scale, the alkaline lysis is normally performed as a batch process. However, this process is not simply scalable. Due to local pH peaks, the nucleic acid might be irreversibly denatured or the extraction is incomplete, both of these effects can result in low product yield or contaminated forms of nucleic acids that are difficult to separate from the intended product.
A further problem of known alkaline lysis techniques is the separation of cellular components. Due to a typical high viscosity of the resulting precipitate, separation can only be performed by pre-filtration or centrifugation and then subsequent clearing filtration. This approach is time consuming and expensive. Furthermore, the DNA (chromosomal DNA as well as plasmid DNA) within the precipitate is sensitive to shearing forces (see Levy, M. S. et al. (2000), Biochemical Engineering Approaches to the Challenges of Producing Pure Plasmid DNA. Trends Biotechnol. 18, 296-305). Therefore, an approach to gently mix the cell lysate and the neutralizing agent in the presence of low shear forces is necessary to avoid contaminating the plasmid containing liquid with chromosomal DNA that can not be easily removed.
In one known extraction procedure, a cell suspension and lysis buffer are pumped into a lysis vessel and the mixing is performed by a stirrer (see Wright, J. L. et al, (2001), Extraction of Plasmid DNA Using Reactor Scale Alkaline Lysis and Selective Precipitation for Scalable Transient Transfection, Cytotechnol. 35, 165-173). After subsequent addition of the neutralizing buffer, the precipitate is then separated from the product (i.e. plasmid) containing liquid. In this process, high concentrations of chromosomal DNA fragments having different sizes are generated due to high shear forces. Such fragments can not easily be separated from the plasmids and are only be detectable via polymerase chain reaction (PCR) analysis.
Another known process requires pumping a cell suspension stream and a lysis buffer stream into a static mixer (see Wan, N. C. et al. (1997), Method for Lysing cells, U.S. Pat. No. 5,837,529). The resulting lysate is then introduced into a second static mixer and mixed with the neutralization buffer. The precipitate generated by this approach must then be separated from the product (plasmid) containing liquid in an expensive and time consuming way, as described above. This technique does avoid any local pH peaks generated through continuous mixing. However, since the shear sensitive neutralization is also performed by a static mixer, the pressure decline over the mixer at this stage in the procedure also leads to shearing of DNA, resulting in a high contamination of the product stream.
In an additional procedure, pH peaks are avoided by mixing small volumes of cell suspension and extraction reagents at a time (see Chevalier, M. (1999), Method and Device for Cell Lysis, CA 0002 31 9021 A1). In this case, mixing is not achieved by using a static mixer but rather by the fast collision of cell suspension and extraction reagents, preferably in stream channels directed towards each other at a right angle. In this process, the diameter of the channel diameter is reduced to 2 from 8 mm, to achieve sufficient mixing. The consequence of this reduction in diameter is that an extracted lysate can only be processed at a maximum speed of 160 mL per minute. Since the neutralization step is also performed in this manner, a high shear force is likewise applied to the precipitated cellular components.
An additional method relates to a non-rigid, gentle mixing of the extracted cell mass with the neutralization buffer, thereby introducing the neutralization buffer into the cell lysate together with air (see Ciccolini, L. A. S. et al. (1999), Rheological Properties of Chromosomal and Plasmid DNA During Alkaline Lysis Reaction. Bioproc. Eng. 21, 231-237).
For the separation of the precipitated cell components, one method utilizes the low density of the flocculated cell components, wherein such components generally float to the surface of the liquid after a relatively long incubation time (see Theodossiou, I. et al. (1999), Methods for Enhancing the Recovery of Plasmid Genes from Neutralised Cell Lysate. Bioproc. Eng. 20, 147-156). However, this process requires longer than one hour due to the only minor difference in the buoyancy of the precipitate compared to the liquid. Thus, the resulting yield using this technique is heavily restricted. Another disadvantage of this method is the necessary and subsequent clear filtration step before any further chromatographic purification of the nucleic acid.
In the present state of the art, there is no known technique whereby the cells are extracted in a gentle way to reduce shearing forces, where the cellular components are separated in a way to avoid contamination of the product stream when the separation of precipitate and product containing liquid stream is rapid. Such a technique would be useful in generating high yields of nucleic acids, without the requirement for any subsequent treatment of the obtained lysate for additional purification (e.g. chromatography or precipitation).